Purified phosphatases are used by molecular biologists to remove phosphate groups from linearized DNA, RNA, nucleotides and proteins in vitro. Removal of both 5′-terminal phosphate residues from a linearized DNA vector prevents recircularization of the vector DNA in a ligation reaction because DNA ligase catalyzes the formation of a phosphodiester bond between adjacent nucleotides only if one nucleotide contains a 5′-phosphate and the other a 3′-hydroxyl group. However, foreign DNA segments with 5′-terminal phosphates can be ligated efficiently into the dephosphorylated vector to give an open circular molecule containing two nicks that can easily be transformed into competent cells.
Several different phosphatases have been developed for removal of phosphate groups from biological molecules, and each has its own advantages and disadvantages. The first phosphatase to be used for this purpose was Bacterial Alkaline Phosphatase (BAP) purified from Escherichia coli (E. coli). BAP has the advantage of having good activity against all types of DNA ends. However, it is difficult to remove from the reaction as it is very resistant to heat and detergents (Sambrook, et al., Molecular Cloning, A Laboratory Manual Sections 1.53-1.72 (1989)). A mutant BAP has been prepared (U.S. Pat. No. 5,891,699, EP Patent No. 0441252) which reportedly had increased thermostability. Calf Intestinal Alkaline Phosphatase (CIP or CIAP) is another phosphatase that has been extensively used in molecular biology techniques (U.S. Pat. Nos. 5,773,226 and 5,707,853). CIAP is not as active on DNA as BAP, but it is slightly easier to remove from a reaction requiring the use of either Proteinase K treatment followed by phenol:chloroform extractions or a heat step in the presence of EDTA followed by a phenol:chloroform extraction (Sambrook, et al., supra (1989)). More recently a phosphatase isolated from Arctic shrimp Pandalus borealis (SAP) (U.S. Pat. Nos. 6,387,634 and 6,379,940) has proved easier to use. It has good activity against all types of DNA ends like BAP, but it is reported to have the advantage that it is easily removed from the reaction by heat inactivation at 65° C. for 15 minutes (Amersham Bioscience, Piscataway, N.J.).
Other reports have occurred in the literature for other thermolabile phosphatases including one purified from a psychrophilic strain TAB5 isolated from Antarctica referred to as Thermolabile Antarctic Phosphatase (TAP) (Rina, et al., Eur. J. Biochem. 267:1230-1238 (2000)). Advantages of TAP over other phosphatases include heat lability and high specific activity. Rina, et al. reported that this phosphatase had a specific activity of 1650 units/mg of protein for p-nitrophenyl phosphate (pNPP) substrate which was significantly higher than the activity of any other known phosphatase. However, the protein produced by the clone reported by Rina, et al., (supra (2000)) had a number of problems associated with overexpression and purification. For example, the purification protocol described by Rina, et al., (supra (2000)) requires multiple ultracentrifugation steps to extract the TAP from cell membranes with which it was apparently associated. This protocol is not suited for large scale manufacture (for example, manufacturing protocols involving production of 300 g or more of cell paste). In addition, overexpressing the TAP gene using the protocol described in Rina, et al., resulted in yields of the enzyme that were very low and consequently not cost effective for large scale manufacture.